CN111175347A

CN111175347A - Preparation method and application of nanowire biosensor

Info

Publication number: CN111175347A
Application number: CN201911370420.8A
Authority: CN
Inventors: 王晗; 孙凯
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-05-19
Anticipated expiration: 2039-12-26
Also published as: WO2021128956A1; CN111175347B

Abstract

The invention relates to a preparation method and application of a nanowire biosensor, and belongs to the technical field of biological tests. The preparation method of the nanowire biosensor can be used for detecting target nucleic acid by designing a corresponding single-stranded nucleic acid probe according to a target to be detected. Meanwhile, continuous dynamic detection of the target nucleic acid can be realized in situ through an electrically heated nucleic acid melting and in-situ release technology, so that the concentration change condition of the target nucleic acid molecule can be mastered. The nanowire biosensor prepared by the method can be used for conveniently and rapidly preparing the required nanowire biosensor and is used for simply and rapidly realizing the melting and in-situ release of the biomarker. The kit can be used for early diagnosis and prognosis of a detected person according to the concentration change of a specific biomarker aiming at the biomarker of a certain disease, and can be widely applied to the fields of tumor early screening and prognosis monitoring, single cell research, embryo culture and development in assisted reproduction and the like.

Description

Preparation method and application of nanowire biosensor

Technical Field

The invention relates to a preparation method and application of a nanowire biosensor, and belongs to the technical field of biological tests.

Background

The real-time detection of the human body biochemical indexes can provide abundant health information with diagnostic value for medical clinical practice, such as human body metabolites, cytokines, biomarkers and other multi-dimensional and multi-level information, wherein the detection of the biomarkers is particularly significant for early screening and prognosis of tumors. At present, the biomarker is mainly obtained by a large-scale medical detection instrument or in-vitro diagnosis equipment through in-vitro analysis of body fluids such as blood, interstitial fluid, urine, excrement and the like and excrement, real-time detection is difficult to realize, and the biomarker has great fall with the requirement of obtaining continuous dynamic health information of a human body. Therefore, it is important to detect the dynamic change trend of the biomarker with high sensitivity and real-time.

The currently used method of high sensitivity nucleic acid detection is an electrobiosensor using Field Effect Transistor (FET) technology as a support. The graphene FET biosensor has good detection performance, a probe which can be used for DNA strand displacement is fixed on the surface of graphene in advance to specifically detect a target fragment, and the graphene FET biosensor has the capability of detecting single nucleotide mutation and can detect Single Nucleotide Polymorphism (SNP). Chinese patent application CN108700535A, entitled nano sensor for nucleic acid detection and identification, discloses a method, a system and a nano sensor device for detecting or identifying nucleic acid with single nucleotide resolution based on nucleic acid strand displacement. The graphene FET field effect transistor used by the detection method is complex in processing flow and long in time consumption, and the processing flow has a graphene transfer printing step, so that the failure rate is high, and the overall processing cost is high. Although the graphene FET field effect transistor sensor has the capability of detecting Single Nucleotide Polymorphism (SNP), if other steps are not needed, only single detection on a completely matched nucleic acid fragment can be realized, repeated measurement on the same nucleic acid fragment for multiple times cannot be realized, and the detection of the dynamic change trend of a target fragment by one sensor cannot be realized.

Disclosure of Invention

The invention aims to provide a preparation method and application of a nanowire biosensor.

The preparation method of the nanowire biosensor provided by the invention has two different preparation methods;

the first preparation method comprises the following steps:

(1) preparing a silicon substrate:

(1-1) repeatedly washing the silicon wafer with acetone and isopropanol, and drying the surface of the silicon wafer with nitrogen;

(1-2) putting the dried silicon wafer into an oxidation furnace, and growing a silicon dioxide insulating layer on the surface of the silicon wafer to obtain a silicon substrate, wherein the thickness of the silicon dioxide insulating layer is 100-1000 nanometers;

(2) preparing a signal acquisition electrode and a metal nanowire on the silicon substrate in the step (1), wherein the method comprises the following steps:

(2-1) preparing a mask chromium plate of the signal acquisition electrode with the alignment mark on the chromium plate by adopting a plate making method for later use;

(2-2) repeatedly washing the silicon substrate in the step (1) with acetone and isopropanol for three times, drying the surface with nitrogen, and placing the silicon substrate on a hot plate at the temperature of 100-120 ℃ for 1-2 minutes to completely dry the silicon substrate;

(2-3) placing the dried silicon substrate on a spin coater, spin-coating electron beam glue on the surface of the silicon substrate, and forming a thin electron beam glue layer with the thickness of 300-400 nanometers on the surface of the silicon substrate;

(2-4) exposing nanowires with the width of 100-500 nanometers and the length of 50-200 micrometers and alignment marks on the central position and the alignment mark position of the silicon substrate spin-coated with the electron beam glue in the step (2-3) by adopting an electron beam exposure method, placing the silicon substrate subjected to electron beam exposure into an electron beam developing solution to remove the electron beam glue on the exposed part, obtaining a nanowire groove and an alignment mark groove on the surface of the silicon substrate, cleaning the nanowire groove and the alignment mark groove by using deionized water, and drying the nanowire groove and the alignment mark groove by using nitrogen;

(2-5) sputtering a metal connecting layer with the thickness of 5-20 nanometers on the silicon substrate with the nano-wire grooves and the alignment mark grooves on the surface in the step (2-4) by adopting a magnetron sputtering method, then sputtering a metal sensing layer with the thickness of 5-300 nanometers, preparing and obtaining metal nanowires and alignment marks on the surface of the silicon substrate, cleaning the metal nanowires and the alignment marks by using a mode of combining acetone and ultrasonic, stripping electron beam glue on the unexposed part of the surface of the silicon substrate from the metal, washing the metal nanowires and the alignment marks by using deionized water, drying the metal nanowires and the alignment marks by using nitrogen, keeping the metal nanowires and the alignment marks on a hot plate at the temperature of 100-;

(2-6) placing the silicon substrate with the metal nanowires and the alignment marks in the step (2-5) on a spin coater, and spin-coating photoresist to form a photoresist thin layer with the thickness of 1.3-1.5 microns on the surface of the silicon substrate; spin coating at 100 rpm for 10 seconds, and then at 4000 rpm for 40 seconds;

(2-7) placing the silicon substrate with the spin-coated photoresist in the step (2-6) on a photoetching machine, placing the mask chromium plate of the signal acquisition electrode in the step (2-1) above the silicon substrate coated with the photoresist, aligning the alignment mark on the silicon substrate with the alignment mark on the mask chromium plate of the signal acquisition electrode, and enabling the silicon substrate and the mask chromium plate of the signal acquisition electrode to be mutually attached, photoetching the silicon substrate from the upper part of the mask chromium plate of the signal acquisition electrode by adopting a photoetching method, then placing the silicon substrate into a developing solution, removing the photoresist on the unexposed part, preparing a signal acquisition electrode groove on the surface of the silicon substrate, cleaning the silicon substrate by using deionized water, and drying the silicon substrate by using nitrogen;

(2-8) carrying out magnetron sputtering on the surface of the silicon substrate with the signal acquisition electrode groove by adopting a magnetron sputtering method, firstly sputtering a metal connecting layer with the thickness of 5-20 nanometers, then sputtering a metal sensing layer with the thickness of 5-300 nanometers, cleaning by adopting a method combining acetone and ultrasound, stripping the photoresist and the metal of the exposed part, washing by using deionized water, drying by using nitrogen, and preparing the signal acquisition electrode and the metal nanowire on the silicon substrate;

(3) preparing a drainage block, wherein the structure of the drainage block is shown in fig. 3, a first through hole and a second through hole are processed in the drainage block, a groove is processed at the bottom of the drainage block, the width of the groove is equal to the length of the metal nanowire in the step (2), the length of the groove is 4-6 mm, the depth of the groove is 50-200 microns, and the groove becomes a bottom channel between the first through hole and the second through hole;

(4) fixing the bottom of the drainage block in the step (3) and the silicon substrate in the step (2) relatively, enabling the length direction of a bottom channel of the drainage block to be vertical to the length direction of the nanowire, and completely covering the nanowire;

(5) connecting a probe on the nanowire in the step (4), wherein the method comprises the following steps:

(5-1) introducing a biotin-modified bovine serum albumin solution dissolved in a phosphate buffer solution into the first through hole of the drainage block, wherein the mass volume concentration of the biotin-modified bovine serum albumin solution is 200 micrograms/ml, so that the first through hole, the bottom channel and the second through hole are filled with the biotin-modified bovine serum albumin solution, the biotin-modified bovine serum albumin solution stays in the bottom channel for 2 hours at room temperature, and the phosphate buffer solution is introduced into the first through hole of the drainage block, so that the biotin-modified bovine serum albumin solution flows out of the second through hole;

(5-2) introducing a streptavidin solution dissolved in phosphate buffer solution into the first through hole of the drainage block, wherein the mass volume concentration of the streptavidin solution dissolved in phosphate buffer solution is 100 micrograms/ml, so that the streptavidin solution dissolved in phosphate buffer solution is filled in the first through hole, the bottom channel and the second through hole, the streptavidin solution dissolved in phosphate buffer solution stays in the bottom channel for 1 hour at 37 ℃, and the phosphate buffer solution is introduced into the first through hole of the drainage block, so that the streptavidin solution dissolved in phosphate buffer solution flows out of the second through hole of the drainage block;

(5-3) introducing a biotin-modified probe solution dissolved in phosphate buffer solution into the first through hole of the drainage block, wherein the molar concentration of the probe solution is 1 micromole/ml, so that the probe solution is filled in the first through hole, the bottom channel and the second through hole, the probe solution stays at room temperature for 1 hour, introducing the phosphate buffer solution into the first through hole of the drainage block, so that the probe solution flows out of the second through hole of the drainage block, and at the moment, the probe is connected with the nanowire to obtain the nanowire biosensor.

Or:

the first preparation method comprises the following steps:

(1) preparing a silicon substrate:

(2-6) placing the silicon substrate with the metal nanowires and the alignment marks in the step (2-5) on a spin coater, spin-coating photoresist to form a photoresist thin layer with the thickness of 1.3-1.5 microns on the surface of the silicon substrate, spin-coating for 10 seconds at 100 revolutions per minute, and then spin-coating for 40 seconds at 4000 revolutions per minute;

(3) preparing a drainage block, processing a first through hole and a second through hole in the drainage block, and processing a groove at the bottom of the drainage block to ensure that the width of the groove is equal to the length of the metal nanowire in the step (2), wherein the length of the groove is 4-6 mm, and the depth of the groove is 50-200 microns, so that the groove becomes a bottom channel between the first through hole and the second through hole;

(5-1) introducing an 11-mercaptoundecanoic acid solution dissolved in pure ethanol into the first through hole of the drainage block, wherein the molar concentration of the 11-mercaptoundecanoic acid solution dissolved in pure ethanol is 1 mmol/L, so that the first through hole, the bottom channel and the second through hole are filled with the 11-mercaptoundecanoic acid solution dissolved in pure ethanol, the 11-mercaptoundecanoic acid solution dissolved in pure ethanol stays in the bottom channel 7 for 1 hour at room temperature, and introducing pure ethanol into the first through hole of the drainage block, so that the 11-mercaptoundecanoic acid dissolved in pure ethanol flows out of the second through hole of the drainage block;

(5-2) introducing a 2- (N-morpholine) ethanesulfonic acid solution having a molar concentration of 50 mmol/l and a pH of 5.0, which contains N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride having a molar concentration of 100 mmol/l and N-hydroxysuccinimide having a molar concentration of 50 mmol/l, into the first through-hole of the flow guide block, filling the first through-hole, the bottom channel, and the second through-hole with the solution, and allowing the solution to stand at room temperature for 30 minutes in the bottom channel 7;

(5-3) introducing a streptavidin solution dissolved in phosphate buffer salt solution into the first through hole of the drainage block, wherein the mass volume concentration of the streptavidin solution dissolved in phosphate buffer salt solution is 100 micrograms/ml, so that the first through hole, the bottom channel and the second through hole are filled with the streptavidin solution, the streptavidin solution stays in the bottom channel 7 for 1 hour at room temperature, and the phosphate buffer salt solution is introduced into the first through hole of the drainage block, so that the streptavidin solution dissolved in phosphate buffer salt solution flows out of the second through hole of the drainage block;

(5-4) introducing a glycine solution dissolved in deionized water into the first through hole of the drainage block, wherein the molar concentration of the glycine solution dissolved in deionized water is 1 mol/L, so that the first through hole, the bottom channel and the second through hole are filled with the glycine solution, the glycine solution stays in the bottom channel 7 for 20 minutes at room temperature, and introducing deionized water into the first through hole of the drainage block, so that the glycine solution dissolved in deionized water flows out of the second through hole of the drainage block;

(5-5) introducing a biotin-modified probe solution dissolved in phosphate buffer solution into the first through hole of the drainage block, wherein the molar concentration of the probe solution is 1 micromole/liter, so that the first through hole, the bottom channel and the second through hole are filled with the probe solution, the probe solution stays in the bottom channel 7 for 1 hour at room temperature, introducing the phosphate buffer solution into the first through hole of the drainage block, so that the probe solution flows out of the second through hole of the drainage block, and at the moment, the probe is connected with the nanowire to obtain the nanowire biosensor.

The second preparation method comprises the following steps:

the method comprises the following steps:

(1) preparing a silicon substrate:

(3) connecting a probe on the nanowire in the step (2), comprising the following steps:

(3-1) respectively placing two leads on the signal acquisition electrode, covering the signal acquisition electrode with the leads by using an insulating substance, and exposing the nanowires to the outside to form a detection unit;

(3-2) placing the detection unit into phosphate buffer saline solution dissolved with biotin-modified bovine serum albumin, wherein the mass volume concentration of the phosphate buffer saline solution dissolved with biotin-modified bovine serum albumin is 200 micrograms/ml, standing for 2 hours at room temperature, taking out the detection unit, washing with the phosphate buffer saline solution, and removing unreacted biotin-modified bovine serum albumin on the surface of the detection unit;

(3-3) putting the detection unit into phosphate buffer solution dissolved with streptavidin, wherein the mass volume concentration of the phosphate buffer solution dissolved with streptavidin is 100 micrograms/ml, standing for 1 hour at 37 ℃, taking out the detection unit, washing with the phosphate buffer solution, and removing unreacted streptavidin on the surface of the detection unit;

(3-4) placing the detection unit into phosphate buffer solution dissolved with biotin-modified probe, keeping the solution at the molar concentration of 1 micromole/ml for one hour at 37 ℃, connecting the probe to the nanowire exposed on the surface of the detection unit, taking out the detection unit, washing with phosphate buffer solution, removing unreacted probe on the surface of the detection unit, and connecting the nanowire to the probe to obtain the nanowire biosensor.

Or:

a second method of preparation comprising the steps of:

(1) preparing a silicon substrate:

(3-2) placing the detection unit into a pure ethanol solution dissolved with 11-mercaptoundecanoic acid, wherein the molar concentration of the pure ethanol solution dissolved with 11-mercaptoundecanoic acid is 1 millimole/liter, standing for 1 hour at room temperature, taking out the detection unit, washing the detection unit with pure ethanol, and removing unreacted 11-mercaptoundecanoic acid on the surface of the detection unit;

(3-3) placing the detecting unit in 2- (N-morpholine) ethanesulfonic acid (MES) having a molar concentration of 50 mmol/l and a pH of 5.0, which contains N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) having a molar concentration of 100 mmol/l and N-hydroxysuccinimide (NHS) having a molar concentration of 50 mmol/l, and allowing the detecting unit to stand at room temperature for 30 minutes, and taking out the detecting unit;

(3-4) quickly placing the detection unit into phosphate buffer solution dissolved with streptavidin, wherein the mass volume concentration of the solution is 100 micrograms/ml, standing for 1 hour at room temperature, taking out the detection unit, washing the detection unit with phosphate buffer solution, and removing the unreacted streptavidin on the surface of the detection unit;

(3-5) putting the detection unit into deionized water with the molar concentration of 1 mol/L and dissolved with glycine, standing for 20 minutes at room temperature, taking out the detection unit, washing the detection unit with the deionized water, and removing unreacted glycine on the surface of the detection unit;

(3-6) placing the detection unit into phosphate buffer solution with the molar concentration of 1 micromole/ml and dissolved with the biotin-modified probe, standing for one hour at 37 ℃, connecting the probe to the nanowire exposed on the surface of the detection unit, taking out the detection unit, washing the detection unit with the phosphate buffer solution, and removing the unreacted probe on the surface of the detection unit to obtain the nanowire biosensor.

The application of the nanowire biosensor prepared by the first method comprises the following steps:

(1) introducing Phosphate Buffered Saline (PBS) into a first through hole of a drainage block in the nanowire biosensor, so that the first through hole, a bottom channel and a second through hole are filled with the PBS, and a first voltage-current curve and an electrochemical impedance spectrum curve of a signal acquisition electrode on the nanowire biosensor are acquired;

(2) introducing a target sequence which is complementary with a probe in the nanowire biosensor into the first through hole of the drainage block, standing for 10 minutes, and collecting a second voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode on the nanowire biosensor;

(3) applying a direct current voltage of 1-1.5 volts to a signal acquisition electrode on the nanowire biosensor for 60-90 seconds to release a target sequence from the nanowire and flow out of a second through hole of the drainage block, and acquiring a third voltage-current curve and an electrochemical impedance spectrum curve of the signal acquisition electrode;

(4) introducing a target sequence which is complementary with a probe in the nanowire biosensor into the first through hole of the drainage block, standing for 10 minutes, and collecting a fourth voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode on the nanowire biosensor;

(5) comparing the first voltage-current curve with the electrochemical impedance spectrum curve, the second voltage-current curve with the electrochemical impedance spectrum curve, the third voltage-current curve with the electrochemical impedance spectrum curve and the fourth voltage-current curve with the electrochemical impedance spectrum curve to realize nucleic acid detection;

(6) repeating the steps (1) to (5) to perform repeated detection of the target nucleic acid sequence.

The application of the nanowire biosensor prepared by the second preparation method comprises the following steps:

(1) putting the nanowire biosensor into a cell culture medium, and collecting a first voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode on the nanowire biosensor;

(2) acquiring a plurality of voltage-current curves and electrochemical impedance spectrum curves of a signal acquisition electrode on the nanowire biosensor according to a set time interval, recording the plurality of voltage-current curves and electrochemical impedance spectrum curves, and comparing the plurality of curves to realize the detection of a target nucleic acid sequence;

(3) applying a direct current voltage of 1-1.5 volts to a signal acquisition electrode on the nanowire biosensor for 60-90 seconds to release a target sequence from the nanowire;

(4) repeating the steps (1) to (3) to detect the target nucleic acid sequence repeatedly for multiple times.

The preparation method and the application of the nanowire biosensor provided by the invention have the advantages that:

the preparation method of the nanowire biosensor provided by the invention has the advantages of convenience, rapidness, low price and the like. The corresponding single-stranded nucleic acid probe is designed according to the detected target, and the target nucleic acid can be detected after the sensor is modified. Then, the nucleic acid melting and in-situ release technology is realized by utilizing the electrical heating characteristic of the nano-wire, and the continuous dynamic detection of the target nucleic acid can be realized in situ, so that the concentration change condition of the target nucleic acid molecule can be mastered. Compared with the traditional melting method, the method has the advantages that the pH value is changed, DNA helicase is used, the temperature is increased, and the like, the nanowire biosensor prepared by the method does not need to add any extra reagent, large auxiliary equipment and the like, and is convenient to miniaturize and carry. The preparation of the nanowire adopts a double-layer structure of a metal connecting layer and a metal sensing layer, the metal connecting layer is made of chromium, titanium and the like, the adhesion between the metal sensing layer and a silicon substrate can be effectively improved, and the metal sensing layer is made of materials with stable chemical properties and excellent electrical properties such as gold and platinum and the like. The sensor is used for detecting a specific biomarker to obtain the concentration change of the biomarker, can be used for carrying out early diagnosis and prognosis on a detected person, can be widely applied to the fields of tumor early screening and prognosis monitoring, single cell research, embryo culture and development in assisted reproduction and the like, and has the advantages of extremely small size, high detection sensitivity and potential development to wearable and implantable equipment.

Drawings

FIG. 1 is a schematic structural view of a nanowire biosensor manufactured by the method of the present invention.

Fig. 2 is a top view of the nanowire biosensor shown in fig. 1.

Fig. 3 is a schematic structural view of a flow guide block in the nanowire biosensor shown in fig. 1.

Fig. 4 is a second structural schematic view of the nanowire biosensor of the present invention.

Fig. 5 is a graph of impedance, conductivity, and time for a nanowire biosensor manufactured according to the present invention without modification.

Fig. 6 is a graph of impedance, conductivity and time after the nanowire biosensor prepared according to the present invention is modified.

Fig. 7 is a graph showing the change of impedance after each operation of the nanowire biosensor manufactured according to the present invention.

Fig. 8 is a graph showing the change in conductivity after each operation of the nanowire biosensor manufactured according to the present invention.

In fig. 1-4, 1 is a silicon substrate, 2 is a flow guide block, 3 is a first through hole, 4 is a signal collecting electrode, 5 is a second through hole, 6 is a metal nanowire, 7 is a flow guide block bottom channel, 8 is a probe, 9 is an alignment mark, 10 is an insulating layer, and 11 is a wire.

Detailed Description

The structure of the nanowire biosensor prepared by the method is shown in fig. 1 and fig. 2, and the preparation method has two types, wherein:

the first preparation method comprises the following steps:

(1) preparation of the silicon substrate 1:

(1-2) putting the dried silicon wafer into an oxidation furnace, and growing a silicon dioxide insulating layer on the surface of the silicon wafer to obtain a silicon substrate, wherein the thickness of the silicon dioxide insulating layer is 100-1000 nanometers, and 500 nanometers is adopted in one embodiment of the invention;

(2) preparing a signal acquisition electrode 4 and a metal nanowire 6 on the silicon substrate 1 in the step (1), wherein the method comprises the following steps:

(2-1) preparing a mask chromium plate with an alignment mark and a signal acquisition electrode on the chromium plate by adopting a plate making (for example, utilizing a direct-writing photoetching device) method for later use;

(2-2) repeatedly washing the silicon substrate 1 in the step (1) with acetone and isopropanol for three times, drying the surface with nitrogen, and placing on a hot plate at 100-120 ℃ for 1-2 minutes to completely dry the silicon substrate 1;

(2-3) placing the dried silicon substrate 1 on a spin coater, spin-coating electron beam glue on the surface of the silicon substrate 1, and forming an electron beam glue thin layer with the thickness of 300-400 nanometers on the surface of the silicon substrate 1, wherein the electron beam glue used in one embodiment of the invention is polymethyl methacrylate;

(2-4) adopting an electron beam exposure method, wherein the acceleration voltage used in one embodiment of the invention is 30keV, the exposure time is 1 minute, the nanowires 6 and the alignment marks 9 with the width of 100-500 nanometers and the length of 50-200 micrometers are exposed at the central position and the alignment mark 9 position of the silicon substrate which is spin-coated with the electron beam glue in the step (2-3), the nanowires 6 and the alignment marks 9 with the width of 200 nanometers and the length of 200 micrometers are exposed in one embodiment of the invention, the silicon substrate after the electron beam exposure is placed into an electron beam developing solution to remove the electron beam glue at the exposed part, the nanowire grooves and the alignment mark grooves are obtained on the surface of the silicon substrate 1, the deionized water is used for cleaning, and the nitrogen;

(2-5) sputtering a metal connecting layer with the thickness of 5-20 nanometers on the silicon substrate with the nanometer wire grooves and the alignment mark grooves on the surface in the step (2-4) by adopting a magnetron sputtering method, sputtering metal chromium with the thickness of 5 nanometers in one embodiment of the invention, then sputtering a metal sensing layer with the thickness of 5-300 nanometers in one embodiment of the invention, sputtering metal gold with the thickness of 100 nanometers in one embodiment of the invention, preparing and obtaining metal nanowires and alignment marks on the surface of the silicon substrate, cleaning by using a mode of combining acetone and ultrasound, stripping electron beam glue and metal on the unexposed part of the surface of the silicon substrate, washing by using deionized water, drying by using nitrogen, keeping on a hot plate at the temperature of 100-120 ℃ for 90 seconds, and completely drying by using the temperature of 120 ℃ in one embodiment of the invention;

(2-6) placing the silicon substrate 1 with the metal nanowires 6 and the alignment marks 9 in the step (2-5) on a spin coater, and spin-coating a photoresist (the photoresist is negative photoresist) to form a photoresist thin layer with the thickness of 1.3-1.5 microns on the surface of the silicon substrate 1; 4340 photoresist was used in one embodiment of the present invention, spin-coated at 100 rpm for 10 seconds, then at 4000 rpm for 40 seconds;

(2-7) placing the silicon substrate with the spin-coated photoresist in the step (2-6) on a photoetching machine, placing the mask chromium plate of the signal acquisition electrode in the step (2-1) above the silicon substrate 1 coated with the photoresist, aligning the alignment mark 9 on the silicon substrate 1 with the alignment mark on the mask chromium plate of the signal acquisition electrode 4, and attaching the silicon substrate 1 and the mask chromium plate of the signal acquisition electrode 4 to each other, photoetching the silicon substrate from the upper side of the mask chromium plate of the signal acquisition electrode 4 by adopting a photoetching method, then placing the silicon substrate into a developing solution, removing the photoresist of the unexposed part, preparing a signal acquisition electrode groove on the surface of the silicon substrate 1, cleaning the silicon substrate with deionized water, and drying the silicon substrate with nitrogen;

(2-8) performing magnetron sputtering on the surface of the silicon substrate with the signal acquisition electrode groove by adopting a magnetron sputtering method, firstly sputtering a metal connecting layer with the thickness of 5-20 nanometers, and then sputtering a metal sensing layer with the thickness of 5-300 nanometers, wherein in one embodiment of the invention, chromium is sputtered on the metal connecting layer with the thickness of 5 nanometers, gold is sputtered on the metal sensing layer with the thickness of 100 nanometers, cleaning is performed by adopting a method combining acetone and ultrasound, photoresist and metal of an exposed part are stripped, washing is performed by using deionized water, drying is performed by using nitrogen, and the signal acquisition electrode 4 and the metal nanowire 6 are prepared on the silicon substrate 1;

(3) preparing a drainage block 2, wherein the structure of the drainage block is as shown in fig. 3, a first through hole 3 and a second through hole 5 are processed in the drainage block 2, a groove is processed at the bottom of the drainage block 1, the width of the groove is equal to the length of the metal nanowire in the step (2), the length of the groove is 4-6 mm, the depth of the groove is 50-200 microns, and the groove is a bottom channel 7 between the first through hole 3 and the second through

hole

5, 5 mm in one embodiment of the invention, and 50 microns in one embodiment of the invention;

(4) and (3) relatively fixing the bottom of the drainage block 2 in the step (3) and the silicon substrate 1 in the step (2), enabling the length direction of a bottom channel 7 of the drainage block 2 to be perpendicular to the length direction of the nanowire 6, and completely covering the nanowire 6, wherein fig. 5 shows curves of impedance, conductivity and time measured by two signal acquisition electrodes 4 after the nanowire biosensor prepared in the step (4) is filled with phosphate buffer solution in the first through hole 3, the bottom channel 7 and the second through hole 5.

(5) Connecting a probe 8 on the nanowire 6 in the step (4), comprising the following steps:

(5-1) introducing a biotin-modified bovine serum albumin solution dissolved in a phosphate buffer solution into the first through hole 3 of the drainage block 2, wherein the mass volume concentration of the biotin-modified bovine serum albumin solution is 200 micrograms/ml, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the biotin-modified bovine serum albumin solution, the biotin-modified bovine serum albumin solution stays in the bottom channel 7 for 2 hours at room temperature, and the phosphate buffer solution is introduced into the first through hole 3 of the drainage block 2, so that the biotin-modified bovine serum albumin solution flows out of the second through hole 5;

(5-2) introducing a streptavidin solution dissolved in phosphate buffer solution into the first through hole 3 of the drainage block 2, wherein the mass volume concentration of the streptavidin solution dissolved in phosphate buffer solution is 100 micrograms/ml, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the streptavidin solution dissolved in phosphate buffer solution, the streptavidin solution dissolved in phosphate buffer solution stays in the bottom channel 7 at 37 ℃ for 1 hour, and the phosphate buffer solution is introduced into the first through hole 3 of the drainage block 2, so that the streptavidin solution dissolved in phosphate buffer solution flows out of the second through hole 5 of the drainage block 2;

(5-3) introducing a biotin-modified probe solution dissolved in phosphate buffer solution into the first through hole 3 of the drainage block 2, wherein the molar concentration of the probe solution is 1 micromole/ml, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the probe solution, standing at room temperature for 1 hour, introducing the phosphate buffer solution into the first through hole 3 of the drainage block 2, so that the probe solution flows out of the second through hole 5 of the drainage block 2, and at the moment, the probe 8 is connected with the upper nanowire 6 to obtain the nanowire biosensor. Fig. 6 is a graph showing the impedance, conductivity and time measured by the two signal collecting electrodes 4 after the nanowire biosensor modified in step (5) is filled with phosphate buffer solution in the first through hole 3, the bottom channel 7 and the second through hole 5.

Or:

a first method of preparation comprising the steps of:

(1) preparation of the silicon substrate 1:

(1-2) putting the dried silicon wafer into an oxidation furnace, and growing a silicon dioxide insulating layer on the surface of the silicon wafer to obtain a silicon substrate 1, wherein the thickness of the silicon dioxide insulating layer is 100-1000 nanometers, and 500 nanometers is adopted in one embodiment of the invention;

(2-2) repeatedly washing the silicon substrate 1 in the step (1) with acetone and isopropanol for three times, drying the surface with nitrogen, and placing on a hot plate at 100-120 ℃ for 1-2 minutes, wherein 120 ℃ is used in one embodiment of the invention to completely dry the silicon substrate;

(2-3) placing the dried silicon substrate on a spin coater, spin-coating electron beam glue on the surface of the silicon substrate, and forming a 300-400 nanometer thick electron beam glue thin layer on the surface of the silicon substrate, wherein the electron beam glue used in one embodiment of the invention is polymethyl methacrylate;

(2-4) adopting an electron beam exposure method, wherein the acceleration voltage used in one embodiment of the invention is 30keV, the exposure time is 1 minute, the nanowires 6 and the alignment marks 9 with the width of 100-500 nanometers and the length of 50-200 micrometers are exposed at the central position and the alignment mark position of the silicon substrate which is spin-coated with the electron beam glue in the step (2-3), the nanowires 6 and the alignment marks 9 with the width of 200 nanometers and the length of 200 micrometers are exposed in one embodiment of the invention, the silicon substrate after the electron beam exposure is placed into an electron beam developing solution to remove the electron beam glue at the exposed part, the nanowire grooves and the alignment mark grooves are obtained on the surface of the silicon substrate 1, the deionized water is used for cleaning, and the nitrogen;

(2-5) sputtering a metal connecting layer with the thickness of 5-20 nanometers on the silicon substrate with the nanometer wire grooves and the alignment mark grooves on the surface in the step (2-4) by adopting a magnetron sputtering method, sputtering metal titanium with the thickness of 5 nanometers in one embodiment of the invention, then sputtering a metal sensing layer with the thickness of 5-300 nanometers in one embodiment of the invention, sputtering metal gold with the thickness of 100 nanometers in one embodiment of the invention, preparing and obtaining the metal nanowire 6 and the alignment mark 9 on the surface of the silicon substrate 1, cleaning by using a mode of combining acetone and ultrasound, stripping electron beam glue and metal on the unexposed part of the surface of the silicon substrate, washing by using deionized water, drying by using nitrogen, keeping on a hot plate at the temperature of 100-120 ℃ for 90 seconds, and completely drying by using the temperature of 120 ℃ in one embodiment of the invention;

(2-7) placing the silicon substrate with the spin-coated photoresist in the step (2-6) on a photoetching machine, placing the mask chromium plate of the signal acquisition electrode in the step (2-1) above the silicon substrate coated with the photoresist, aligning the alignment mark 9 on the silicon substrate 1 with the alignment mark on the mask chromium plate of the signal acquisition electrode, and enabling the silicon substrate 1 and the mask chromium plate of the signal acquisition electrode to be mutually attached, photoetching the silicon substrate from the upper part of the mask chromium plate of the signal acquisition electrode by adopting a photoetching method, then placing the silicon substrate into a developing solution, removing the photoresist of the unexposed part, preparing a signal acquisition electrode groove on the surface of the silicon substrate 1, cleaning the silicon substrate with deionized water, and drying the silicon substrate with nitrogen;

(2-8) performing magnetron sputtering on the surface of the silicon substrate with the signal acquisition electrode groove by adopting a magnetron sputtering method, firstly sputtering a metal connecting layer with the thickness of 5-20 nanometers, and then sputtering a metal sensing layer with the thickness of 5-300 nanometers, wherein in one embodiment of the invention, titanium on the metal connecting layer with the thickness of 5 nanometers is sputtered, then gold on the metal sensing layer with the thickness of 100 nanometers is sputtered, cleaning is performed by adopting a method combining acetone and ultrasonic, the photoresist of the exposed part is stripped from the metal, washing is performed by using deionized water, drying is performed by using nitrogen, and the signal acquisition electrode 4 and the metal nanowire 6 are prepared on the silicon substrate;

(3) preparing a drainage block 2, processing a first through hole 3 and a second through hole 5 in the drainage block 2, and processing a groove at the bottom of the drainage block 2 to ensure that the width of the groove is equal to the length of the metal nanowire in the step (2), the length of the groove is 4-6 mm, and the depth of the groove is 50-200 microns, so that the groove becomes a bottom channel 7 between the first through hole and the second through hole, wherein the length of the groove is 5 mm in one embodiment of the invention, and the depth of the groove is 50 microns in the embodiment of the invention;

(4) fixing the bottom of the drainage block 2 in the step (3) and the silicon substrate 1 in the step (2) relatively, so that the length direction of a bottom channel 7 of the drainage block 2 is perpendicular to the length direction of the nanowire 6, and the nanowire 6 is completely covered;

(5-1) introducing an 11-mercaptoundecanoic acid solution dissolved in pure ethanol into the first through hole 3 of the drainage block 2, wherein the molar concentration of the 11-mercaptoundecanoic acid solution dissolved in pure ethanol is 1 mmol/l, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the 11-mercaptoundecanoic acid solution dissolved in pure ethanol, the 11-mercaptoundecanoic acid solution dissolved in pure ethanol stays in the bottom channel 7 for 1 hour at room temperature, and introducing pure ethanol into the first through hole 3 of the drainage block 2, so that the 11-mercaptoundecanoic acid dissolved in pure ethanol flows out of the second through hole 5 of the drainage block 2;

(5-2) introducing a 2- (N-morpholine) ethanesulfonic acid solution having a molar concentration of 50 mmol/l and a pH of 5.0, which contains N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) having a molar concentration of 100 mmol/l and N-hydroxysuccinimide (NHS) having a molar concentration of 50 mmol/l, into the first through-hole 3 of the drainage block 2, filling the first through-hole 3, the bottom channel 7, and the second through-hole 5 with the solution, and allowing the solution to stand at room temperature for 30 minutes in the bottom channel 7;

(5-3) introducing a streptavidin solution dissolved in phosphate buffer solution into the first through hole 3 of the drainage block 2, wherein the mass volume concentration of the streptavidin solution dissolved in phosphate buffer solution is 100 micrograms/ml, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the streptavidin solution, the streptavidin solution stays in the bottom channel 7 for 1 hour at room temperature, and introducing phosphate buffer solution into the first through hole 3 of the drainage block 2, so that the streptavidin solution dissolved in phosphate buffer solution flows out of the second through hole 5 of the drainage block 2;

(5-4) introducing a glycine solution dissolved in deionized water into the first through hole 3 of the drainage block 2, wherein the molar concentration of the glycine solution dissolved in deionized water is 1 mol/L, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the solution, the solution stays in the bottom channel 7 for 20 minutes at room temperature, and introducing deionized water into the first through hole 3 of the drainage block 2, so that the glycine solution dissolved in deionized water flows out of the second through hole 5 of the drainage block 2;

(5-5) introducing a biotin-modified probe solution dissolved in phosphate buffer solution into the first through hole 3 of the drainage block 2, wherein the molar concentration of the probe solution is 1 micromole/liter, so that the first through hole 3, the bottom channel 7 and the second through hole 5 are filled with the probe solution, the probe solution stays in the bottom channel 7 for 1 hour at room temperature, the phosphate buffer solution is introduced into the first through hole 3 of the drainage block 2, the probe solution flows out of the second through hole 5 of the drainage block 2, and at the moment, the probe 8 is connected with the upper nanowire 6 to obtain the nanowire biosensor.

The second method of the present invention is a nanowire biosensor, the structure of which is shown in fig. 4, and the second method comprises the following steps:

(1) preparation of the silicon substrate 1:

(2) preparing a signal acquisition electrode 4 and a metal nanowire 6 on the silicon substrate in the step (1), wherein the method comprises the following steps:

(2-4) adopting an electron beam exposure method, wherein the acceleration voltage used in one embodiment of the invention is 30keV, the exposure time is 1 minute, nanowires and alignment marks with the width of 100-500 nanometers and the length of 50-200 micrometers are exposed at the central position and the alignment mark position of the silicon substrate which is spin-coated with the electron beam glue in the step (2-3), the nanowires and the alignment marks are 200 nanometers wide and 200 micrometers long in one embodiment of the invention, the silicon substrate after the electron beam exposure is placed into an electron beam developing solution to remove the electron beam glue at the exposed part, a nanowire groove and an alignment mark groove are obtained on the surface of the silicon substrate, the nanowire groove and the alignment mark groove are cleaned by deionized water, and nitrogen is dried;

(2-5) sputtering a metal connecting layer with the thickness of 5-20 nanometers on the silicon substrate with the nanometer wire grooves and the alignment mark grooves on the surface in the step (2-4) by adopting a magnetron sputtering method, sputtering metal chromium with the thickness of 5 nanometers in one embodiment of the invention, then sputtering a metal sensing layer with the thickness of 5-300 nanometers in one embodiment of the invention, sputtering metal platinum with the thickness of 100 nanometers in one embodiment of the invention, preparing and obtaining the metal nanowire 6 and the alignment mark 9 on the surface of the silicon substrate 1, cleaning by using a mode of combining acetone and ultrasound, stripping electron beam glue and metal on the unexposed part of the surface of the silicon substrate, washing by using deionized water, drying by using nitrogen, keeping on a hot plate at the temperature of 100-120 ℃ for 90 seconds, and completely drying by using the temperature of 120 ℃ in one embodiment of the invention;

(2-6) placing the silicon substrate 1 with the metal nanowires 6 and the alignment marks 9 in the step (2-5) on a spin coater, and spin-coating a photoresist (the photoresist is negative photoresist) to form a photoresist thin layer with the thickness of 1.3-1.5 microns on the surface of the silicon substrate; 4340 photoresist was used in one embodiment of the present invention, spin-coated at 100 rpm for 10 seconds, then at 4000 rpm for 40 seconds;

(2-7) placing the silicon substrate with the spin-coated photoresist in the step (2-6) on a photoetching machine, placing the mask chromium plate of the signal acquisition electrode in the step (2-1) above the silicon substrate 1 coated with the photoresist, aligning the alignment mark 9 on the silicon substrate 1 with the alignment mark on the mask chromium plate of the signal acquisition electrode, and attaching the silicon substrate 1 and the mask chromium plate of the signal acquisition electrode to each other, photoetching the silicon substrate from the upper side of the mask chromium plate of the signal acquisition electrode by adopting a photoetching method, then placing the silicon substrate into a developing solution, removing the photoresist at the unexposed part, preparing a signal acquisition electrode groove on the surface of the silicon substrate 1, cleaning the silicon substrate with deionized water, and drying the silicon substrate with nitrogen;

(2-8) performing magnetron sputtering on the surface of the silicon substrate with the signal acquisition electrode groove by adopting a magnetron sputtering method, firstly sputtering a metal connecting layer with the thickness of 5-20 nanometers, and then sputtering a metal sensing layer with the thickness of 5-300 nanometers, wherein in one embodiment of the invention, a metal connecting layer chromium with the thickness of 5 nanometers is sputtered, then a metal sensing layer platinum with the thickness of 100 nanometers is sputtered, cleaning is performed by adopting a method combining acetone and ultrasound, the photoresist of the exposed part is stripped from the metal, washing is performed by using deionized water, drying is performed by using nitrogen, and the signal acquisition electrode 4 and the metal nanowire 6 are prepared on the silicon substrate 1;

(3) connecting a probe 8 on the nanowire 6 in the step (2), comprising the following steps:

(3-1) respectively placing two leads 11 on the signal collecting electrode 4, covering the signal collecting electrode 4 with the leads by using an insulating substance 10, and exposing the nanowire 6 to the outside to form a detection unit;

Or:

the second preparation method comprises the following steps:

(1) preparation of the silicon substrate 1:

(2-1) preparing a mask chromium plate of the signal acquisition electrode with the alignment mark on the chromium plate by adopting a plate making (direct writing photoetching equipment) method for later use;

(2-4) adopting an electron beam exposure method, wherein the acceleration voltage used in one embodiment of the invention is 30keV, the exposure time is 1 minute, nanowires and alignment marks with the width of 100-500 nanometers and the length of 50-200 micrometers are exposed at the central position and the alignment mark position of the silicon substrate which is spin-coated with the electron beam glue in the step (2-3), the nanowires and the alignment marks are 200 nanometers wide and 200 micrometers long in one embodiment of the invention, the silicon substrate after the electron beam exposure is placed into an electron beam developing solution to remove the electron beam glue at the exposed part, a nanowire groove and an alignment mark groove are obtained on the surface of the silicon substrate 1, the nanowire groove and the alignment mark groove are cleaned by deionized water, and nitrogen is dried;

(2-5) sputtering a metal connecting layer with the thickness of 5-20 nanometers on the silicon substrate with the nanometer wire grooves and the alignment mark grooves on the surface in the step (2-3) by adopting a magnetron sputtering method, sputtering metal titanium with the thickness of 5 nanometers in one embodiment of the invention, then sputtering a metal sensing layer with the thickness of 5-300 nanometers in one embodiment of the invention, sputtering metal platinum with the thickness of 100 nanometers in one embodiment of the invention, preparing and obtaining the metal nanowire 6 and the alignment mark 9 on the surface of the silicon substrate 1, cleaning by using a mode of combining acetone and ultrasound, stripping electron beam glue and metal on the unexposed part of the surface of the silicon substrate, washing by using deionized water, drying by using nitrogen, keeping on a hot plate at the temperature of 100-120 ℃ for 90 seconds, and completely drying by using the temperature of 120 ℃ in one embodiment of the invention;

(2-8) performing magnetron sputtering on the surface of the silicon substrate with the signal acquisition electrode groove by adopting a magnetron sputtering method, sputtering a metal connecting layer with the thickness of 5-20 nanometers firstly, and then sputtering a metal sensing layer with the thickness of 5-300 nanometers, sputtering metal titanium with the thickness of 5 nanometers in one embodiment of the invention, then sputtering metal platinum with the thickness of 100 nanometers, cleaning by adopting a method combining acetone and ultrasonic, stripping the exposed photoresist and metal, washing with deionized water, drying with nitrogen, and preparing a signal acquisition electrode 4 and a metal nanowire 6 on the silicon substrate;

(1) introducing Phosphate Buffered Saline (PBS) into a first through hole 3 of a drainage block 2 in the nanowire biosensor to enable the phosphate buffered saline to fill the first through hole 3, a bottom channel 7 and a second through hole 5, and connecting a first voltage-current curve and an electrochemical impedance spectrum curve of a signal acquisition electrode 4 on the signal acquisition nanowire biosensor by using an electrochemical workstation;

(2) introducing a target sequence which is complementary with a probe in the nanowire biosensor into the first through hole 3 of the drainage block 2, standing for 10 minutes, and collecting a second voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode 4 on the nanowire biosensor;

(3) applying a direct current voltage of 1-1.5 volts to a signal acquisition electrode 4 on the nanowire biosensor for 60-90 seconds, wherein 1.2 volts is used in one embodiment of the invention for 60 seconds, so that a target sequence is released from the nanowire 6 and flows out of a second through hole 5 of a drainage block 2, and a third voltage-current curve and an electrochemical impedance spectroscopy curve of the signal acquisition electrode 4 are acquired; fig. 7 shows the change curve of the impedance measured by the two signal collecting electrodes 4 after each step of the modification operation and each step of the above (1) to (3) of the nanowire biosensor prepared by the first method. Fig. 8 shows a change curve of the conductivity measured by the two signal collecting electrodes 4 after each modification operation and each operation of the above-mentioned (1) to (3) of the nanowire biosensor manufactured by the first method. In fig. 7 and 8, 1 represents data measured by two signal collecting electrodes 4 in an initial state of the nanowire biosensor, 2 represents data measured by two signal collecting electrodes 4 after the nanowire biosensor is modified by biotin-modified bovine serum albumin, 3 represents data measured by two signal collecting electrodes 4 after the nanowire biosensor is modified by streptavidin, 4 represents data measured by two signal collecting electrodes 4 after the nanowire biosensor is modified by a biotin-modified probe, 5 represents data measured by two signal collecting electrodes 4 after 1 femtomole target sequence is introduced into the nanowire biosensor and stands for 15 minutes, 6 represents data measured by two signal collecting electrodes 4 after phosphate buffer solution is introduced into the nanowire biosensor and stands for 15 minutes after 1 picomole target sequence is introduced into the nanowire biosensor, after phosphate buffer solution is introduced, data measured by the two signal acquisition electrodes 4 are represented by 7, 1.1-volt direct-current voltage is applied to the two signal acquisition electrodes 4, after the application time is 1 minute, the data measured by the two signal acquisition electrodes 4 are represented by 8, 1 picomolar target sequence is introduced into the nanowire biosensor and stands still for 15 minutes, and after the phosphate buffer solution is introduced, the data measured by the two signal acquisition electrodes 4 are represented by 8.

(4) Introducing a target sequence which is complementary with a probe in the nanowire biosensor into the first through hole 3 of the drainage block 2, standing for 10 minutes, and collecting a fourth voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode 4 on the nanowire biosensor;

The application of the nanowire biosensor prepared by the second method comprises the following steps:

(1) putting the nanowire biosensor into a cell culture medium, and collecting a first voltage-current curve and an electrochemical impedance spectrum curve of a signal collecting electrode 4 on the nanowire biosensor;

The principle of the method is that in the prior art, the chain scission method comprises heating, pH value change, helicase use and the like, and the heating is the best scheme for realizing nucleic acid melting without adding additional reagents. Metal nanowires meet the above requirements and have therefore been selected as the core component of the sensor. The metal nanowire is prepared in a double-layer mode, the lower layer is a metal connecting layer, common metals are chromium, titanium and the like, the upper layer is a metal sensing layer, and the common metals are gold, platinum and the like. The metal has excellent physicochemical properties, can not generate chemical reaction with most chemicals, and has good stability. Gold and platinum nanostructures are excellent nano-electrode candidates in electrochemical applications, and can be applied to pressure sensors, DNA detection sensors and the like. Meanwhile, the metal nanowire can provide high current density, high signal-to-noise ratio and low double-electron-layer capacitance, and is very suitable for being manufactured into a sensor.

Due to the excellent conductivity and heat conduction performance of the metal nanowire, the width of the metal nanowire is in a nanometer level, the length of the metal nanowire is in a micrometer level, after a fixed voltage is applied to two ends of the metal nanowire for a certain time, a certain temperature can be generated on the surface of the metal nanowire, the captured biomarker can be used for melting and in-situ releasing, and meanwhile, the fact that the modification performed on the surface of the metal nanowire cannot be damaged is guaranteed.

The modification system used in the present invention is based on the binding of avidin to biotin. According to literature research, the bond energy of adenine and thymine is 8kJ/mol or 1.9kcal/mol, and the bond energy of guanine and cytosine is 13kJ/mol or 3.1 kcal/mol. The affinity constant of avidin combined with biotin is million times of that of common antigen-antibody reaction, the dissociation constant of the complex formed by the two is very small, the avidin and the biotin cannot be denatured at 100 ℃ and present irreversible reactivity, and the combination of acid, alkali, a denaturant, proteolytic enzyme and an organic solvent is not influenced.

Therefore, in the process that the metal nanowire generates a large amount of heat by applying an electric pulse signal to the nanowire, the combination stability of the avidin and the biotin is far higher than that of a DNA double strand and an antigen-antibody reaction, so that the melting and in-situ release of the biomarker can be realized by accurately controlling the surface heating of the metal nanowire. The method is used for dynamic real-time detection of the content of the biomarkers to reflect the physical health condition of a patient.

Claims

1. A method for preparing a nanowire biosensor, the method comprising the steps of:

(1) preparing a silicon substrate:

(2-6) placing the silicon substrate with the metal nanowires and the alignment marks in the step (2-5) on a spin coater, and spin-coating a photoresist (the photoresist is negative photoresist) to form a photoresist thin layer with the thickness of 1.3-1.5 microns on the surface of the silicon substrate; 4340 photoresist was used in one embodiment of the present invention, spin-coated at 100 rpm for 10 seconds, then at 4000 rpm for 40 seconds;

2. A method for preparing a nanowire biosensor, the method comprising the steps of:

(1) preparing a silicon substrate:

3. A method for preparing a nanowire biosensor, the method comprising the steps of:

(1) preparing a silicon substrate:

4. A method for preparing a nanowire biosensor, the method comprising the steps of:

(1) preparing a silicon substrate:

5. The method of claim 1, 2, 3 or 4, wherein the metal connection layer in the step (2-8) is chromium or titanium, and the metal sensing layer is gold or platinum.

6. Use of a nanowire biosensor according to claims 1 and 2, characterized in that the use comprises the following steps:

7. Use of a nanowire biosensor according to claims 3 and 4, characterized in that the use comprises the following steps: